Solid culture medium and method for preparing the same

ABSTRACT

A solid medium having a 10 minute-average water absorption rate of at least 0.05 ml/minute, which is obtainable by a method for producing a solid medium comprising the steps of dissolving components of the solid medium other than solvent water into the solvent water, solidifying the obtained solution, and drying the solidified medium to remove water, wherein water is removed in such an amount that the solid medium after the removal of water should have the 10 minute-average water absorption rate of at least 0.05 ml/minute, and the amount of the solvent water is larger than a prescribed amount by an amount almost equal to the amount of the water to be removed. The solid medium does not cause growth inhibition of microorganisms due to drying, shows a superior water absorption rate to enable application of a larger amount of a sample in a short period of time, and is suitable for quick and accurate measurement tests of microbial numbers.

RELATED APPLICATIONS

This application is the U.S. National Phase under 35U.S.C. §371 ofInternational Application PCT/JP 01/06872, filed Aug. 9, 2001, which waspublished in a language other than English, which claims priority ofJapanese Patent Application No. 2001-20606, filed Jan. 29, 2001 all ofwhich are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a solid medium used for growth andtests of various microorganisms and so forth and a method for producingthe same.

BACKGROUND ART

As an example of dried solid media, a dried solid medium isconventionally known which is prepared by lyophilizing a medium pouredinto a plastic dish and solidified (JP 60-19988B, referred to as “Priorart 1” hereinafter).

Further, there is also a known dried solid medium in the form of a film,which contains a gelling agent such as agar and is easily substantiallyrestored to a previous state before it is formed into film by addingsterilized water (JP 6-311880A, referred to as “Prior art 2”hereinafter).

However, these conventional materials have the following problems.

Prior art 1 and Prior art 2 mentioned above are used in order to improvestorage stability of media and the media are dried so that a watercontent in the compositions of finally obtained solid medium becomes 50%or less. Therefore, they have a problem that they require a long periodof time as long as about 3 hours for full restoration of the media uponuse.

Further, since the dried solid media obtained according to theaforementioned conventional techniques require a long period of time forrestoration as described above, restoration in a relatively short periodsuch as 1 to 10 minutes results in insufficient restoration. If suchmedia are used for microorganism tests, growth of Staphylococcus aureusATCC 6538P, Bacillus stearothermophilus var. calidolactis NIZO C953etc., which are important bacterial species in microorganism tests offoodstuffs and so forth, is partially inhibited due to the insufficientrestoration of the medium, resulting in inaccurate measurement of thenumber of microorganisms, as demonstrated by the Test Examples mentionedlater.

That is, the conventional dried solid media have a problem that theyrequire a long period of time for restoration, and thus they are notsuitable for quick and accurate measurement tests of microbial numbers.

Further, it is known that a solid medium is dried in order to removeexcessive moisture on its surface after its production. Since thisdrying process removes a part of solvent water from the solid mediumprepared in a prescribed composition, water content of the medium uponactual culture of microorganisms should significantly differ from theprescribed content in the composition identifying the solid medium.Influences of medium-drying on the culture of microorganisms cannot beignored. For example, it is known that growth of microorganisms issometimes degraded due to drying etc., even when a medium is stored in atest tube with a cotton plug for several weeks. Moreover, uncontrolledremoval of water sometimes makes the composition of solid mediainconstant. Therefore, reproducible and accurate results may not beobtained in culture tests of microorganisms, especially those ofmicroorganisms that are likely to be affected by the water content ofmedium.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a solid medium whichshows a superior water absorption rate to enable application of a largeamount of sample in a short period of time and is suitable for quick andaccurate measurement tests of microbial numbers and to provide a methodfor producing the same.

The inventors of the present invention assiduously studied in view ofthe aforementioned problems of the prior art. As a result, they obtaineda solid medium by dissolving components of the solid medium into thesolvent water whose amount is larger than a prescribed amount,solidifying the obtained solution and drying the solidified medium toremove water in an amount almost equal to the excessive amount of thesolvent water. They have found that the medium does not cause growthinhibition of microorganisms due to drying. They have also found that itshows superior water absorption rate to enable application of a largeamount of a sample in a short period of time and is suitable for quickand accurate measurement tests of microbial numbers. Thus, theyaccomplished the present invention.

The present invention provides a solid medium having a 10 minute-averagewater absorption rate of at least 0.05 ml/minute (also referred to asthe “solid medium of the present invention” hereinafter), which isobtainable by a method for producing a solid medium comprising the stepsof dissolving components of the solid medium other than solvent waterinto the solvent water, solidifying the obtained solution, and dryingthe solidified medium to remove water, wherein water is removed in suchan amount that the solid medium after the removal of water should havethe 10 minute-average water absorption rate of at least 0.05 ml/minute,and the amount of the solvent water is larger than a prescribed amountby an amount almost equal to the amount of the water to be removed.

In the solid medium of the present invention, the amount of the water tobe removed is preferably at least 5% of the solvent water, morepreferably at least 30% of the solvent water.

The solid medium of the present invention preferably has a water contentof at least 90%.

The present invention also provides a method for producing a solidmedium comprising the steps of dissolving components of the solid mediumother than solvent water into the solvent water, solidifying theobtained solution and drying the solidified medium to remove water,wherein water is removed in such an amount that the solid medium afterthe removal of water should have a 10 minute-average water absorptionrate of at least 0.05 ml/minute, and the amount of the solvent water islarger than a prescribed amount by an amount almost equal to the amountof the water to be removed.

In the production method of the present invention, the amount of thewater to be removed is preferably at least 5% of the solvent water, morepreferably at least 30% of the solvent water.

In the present specification, a solid medium means a medium that is in asolid state at the time of use, for example, a plate medium, slantmedium and so forth, which are solidified with a gelling agent such asagar.

A medium is generally identified (defined) by its composition. Thus, theprescribed amount of solvent water is an amount of water in such acomposition used for the identification of the medium. The prescribedamount of solvent water means an amount of water added in order todissolve each component of the solid medium other than solvent water,and when a component of the solid medium is specified as an aqueoussolution, the amount of water in this component is not included.

The 10 minute-average water absorption rate is calculated in accordancewith the following equation by using A and B measured as follows. Totalweight (B) of a sample plate (circular shape, outer diameter: 9 cm,inner diameter: 8.6 cm (area: 58.1 cm²), amount of medium: 15 g) ismeasured, and water absorption is started by adding 10 ml ofion-exchanged water onto the plate medium. After 10 minutes, water thathas not been absorbed is discarded, moisture on a dish wall is wipedoff, and the total weight (A) of the sample plate after the waterabsorption is measured. The measurement is performed at 25° C. When thearea of the sample plate differs from the aforementioned area, the testis performed by using ion exchanged water in an amount of 10 ml perabove-defined area, and the result is converted into a water absorptionrate per above-mentioned area.Water absorption rate (ml/minute)=(A−B)/10

The amount of water removed by drying is calculated by measuring theweight loss due to drying. That is, the weight loss due to drying, i.e.,so-called water loss (c), can be obtained from the medium weight beforethe drying (a) and the medium weight after the drying (b) in accordancewith an equation of a−b=c. Then, a ratio of the removed water, i.e.,percentage of water removed by drying (e, %) can be obtained from thetotal amount of added solvent water (d) and the aforementioned weightloss due to drying (c) in accordance with the following equation.e(%)=c/d×100

A water content of solid medium means a percentage of water content (%)defined as (amount of water in solid medium)/(amount of solidcontent+amount of water)×100. Here, both the amount of water and thesolid content are represented in weight.

BEST MODE FOR CARRYING OUT THE INVENTION

A medium is a nutriment for growing or proliferating microorganisms suchas bacteria, yeasts and molds, and it is also their growing environment.A medium usually contains, as medium components, a saccharide such asglucose and lactose, a nitrogen source such as amino acids, peptone,nitrates and ammonium salts, inorganic salts such as those of potassium,phosphorus and magnesium, a growth factor such as vitamins and so forth.Media are roughly classified into liquid media in which mediumcomponents are simply dissolved in solvent water and solid mediasolidified by addition of a gelling agent into liquid media. The presentinvention provides a solid medium, which is, as defined above, a medium(culture medium) in a solid state at the time of use, for example, aplate medium, slant medium or the like, produced by solidifying with agelling agent such as agar.

Examples of the gelling agent include agar, gelatin, gellan gum,carrageenan and so forth.

Specific examples of the composition of the solid medium of the presentinvention include the compositions of the followings: the nutrient agarmedium, the standard agar medium, the deoxycholate agar medium, theE.M.B. medium, the Endo medium, the plate count agar medium with B.C.P.,the mannitol salt agar with egg yolk, the potato dextrose agar medium,the Violet red bile lactose (VRBL) agar medium, the yeastextract-glucose-chloramphenicol agar medium, the Acidified MRS medium,Medium M17, the Baird-Parker agar medium (ETGP agar medium) and soforth, which are defined in microorganism test methods described in theJapanese Food Sanitation Law, the Japan Pharmacopoeia, the InternationalDiary Federation Standard (IDF STANDARD) and so forth [“NyuseihinShikenhou Chukai (Commentary of Test Methods of Dairy Products)”, Ed. bythe Pharmaceutical Society of Japan, pp.111–125, Apr. 10, 1990, KaneharaShuppan Co., Ltd. (Reference 1); “IDF STANDARD (Revised Version of1991)”, p.306, p.470, pp.645–647, Dec. 25, 1991, published byInternational Dairy Federation of Japan (Reference 2); “Shin SaikinBaichi-gaku Koza Ge II (Lecture of Culture Medium Science for Bacteria,Vol. 2, II)”, Second edition, Editorship: Sakazaki Toshikazu, pp.62–63,Aug. 15, 1996, Kindai Shuppan Co., Ltd. (Reference 3)).

For reference, the prescribed amounts of respective components containedin the aforementioned solid media described in Reference 1 are mentionedbelow.

Prescribed amounts of components in the solid medium composition of thenutrient agar medium (pH 7.0 to 7.4) are 5 g of meat extract, 10 g ofpeptone, 1 to 2 g of sodium chloride (NaCl), 12 to 15 g of agar and 1000ml (1 liter) of purified water (solvent water ).

Prescribed amounts of components in the solid medium composition of thestandard agar medium (pH 6.8 to 7.2) are 2.5 g of yeast extract, 5 g ofpeptone, 1 g of glucose, 15 g of agar and 1000 ml (1 liter) of purifiedwater (solvent water).

Prescribed amounts of each component in the solid medium composition ofthe deoxycholate agar medium (pH 7.0 to 7.4) are 10 g of peptone, 10 gof lactose, 1 g of sodium deoxycholate, 5 g of sodium chloride (NaCl), 2g of K₂HPO₄, 0.033 g of Neutral Red, 2 g of ferric ammonium citrate, 15g of agar and 1000 ml (1 liter) of purified water (solvent water).

Prescribed amounts of components in the solid medium composition of theE.M.B. medium (pH 6.6 to 7.0) are 10 g of peptone, 10 g of lactose, 2 gof K₂HPO₄, 0.4 g of Eosin Y, 0.065 g of Methylene Blue, 18 g of agar and1000 ml (1 liter) of purified water (solvent water).

Prescribed amounts of components in the solid medium composition of theEndo medium (pH 7.0 to 7.4) are 10 g of peptone, 3 g of meat extract, 10g of lactose, 1.6 g of Na₂SO₃, 0.1 g of Basic Fuchsine, 15 g of agar and1000 ml (1 liter) of purified water (solvent water).

Prescribed amounts of components in the solid medium composition of theplate count agar medium with B.C.P. (pH 6.0 to 7.0) are 2.5 g of yeastextract, 5 g of peptone, 1 g of glucose, 1 g of Tween 80, 0.1 g ofL-cysteine, 0.06 g of Bromocresol Purple, 15 g of agar and 1000 ml (1liter) of purified water (solvent water).

Prescribed amounts of components in the solid medium composition of themannitol salt agar with egg yolk [pH 7.2 to 7.6, where 50 to 60ml offresh egg yolk solution (obtained by dissolving egg yolk into the sameamount of physiological saline) is added after sterilization with highpressure steam and before pouring the medium into plate] are 2.5 g ofmeat extract, 10 g of peptone, 10 g of mannitol, 75 g of sodium chloride(NaCl), 0.025 g of Phenol Red, 15 g of agar and 1000 ml (1 liter) ofpurified water (solvent water).

Prescribed amounts of components in the solid medium composition of thepotato dextrose agar (pH 5.6 to 5.7) are 200 g of potato infusion, 20 gof glucose, 15 g of agar and 1000 ml (1 liter) of purified water(solvent water).

Prescribed amounts of respective components contained in the solid mediadescribed in Reference 2 are mentioned hereafter.

Prescribed amounts of components in the solid medium composition of theViolet red bile salt lactose (VRBL) agar medium (pH 7.4±0.1) are 7 g ofpeptone, 3 g of yeast extract, 10 g of lactose (C₁₂H₂₂O₁₁.H₂O), 5 g ofsodium chloride (NaCl), 1.5 g of bile acid salt, 0.03 g of Neutral Red,0.002 g of Crystal Violet, 12 to 18 g of agar and 1000 ml (1 liter) ofwater (solvent water).

Prescribed amounts of components in the solid medium composition of theyeast extract-glucose-chloramphenicol agar medium (pH 6.6) are 5 g ofyeast extract, 20 g of glucose (C₆H₁₂O₆), 0.1 g of chloramphenicol(C₁₁H₁₂Cl₂N₂O₅) or oxytetracycline (C₂₂H₃₀N₂O₁₁), 12 to 15 g of agar and1000 ml (1 liter) of water (solvent water).

Prescribed amounts of components in the solid medium composition of theacidified MRS medium (adjusted to pH 5.4 with acetic acid) are 10 g ofPeptone 1 (trypsin-digested product of casein), 10 g of meat extract, 5g of yeast extract (powder), 20 g of glucose (C₆H₁₂O₆), 1 ml of Tween 80(sorbitan mono-oleate), 2 g of dipotassium hydrogenorthophosphate(K₂HPO₄), 5 g of sodium acetate trihydrate (CH₃CO₂Na.3H₂O), 2 g ofdiammonium citrate (C₆H₆O₇(NH₄)₂), 0.2 g magnesium sulfate heptahydrate(MgSO₄.7H₂O), 0.05 g of manganese sulfate tetrahydrate (MnSO₄.4H₂O), 9to 18 g of agar and 1000 ml (1 liter) of water (solvent water).

Prescribed amounts of components in the solid medium composition ofMedium M17 [pH 7.1 to 7.2, where lactose (C₁₂H₂₂O₁₁) is added as asterilized 10% lactose (C₁₂H₂₂O₁₁) aqueous solution after sterilizationwith high pressure steam and before pouring the medium into plate] are2.50 g of Peptone 1 (trypsin-digested product of casein), 2.50 g ofPeptone 2 (pepsin-digested product of meat), 5.00 g of Peptone 3 (papain-digested product of soybean), 2.50 g of yeast extract (powder), 5.00 gof meat extract, 19.00 g of β-glycerophosphoric acid salt (disodiumsalt, C₆H₇O₆PNa₂), 0.25 g of magnesium sulfate heptahydrate(MgSO₄.7H₂O), 0.50 g of ascorbic acid (C₆H₈O₆), 5 g of lactose(C₁₂H₂₂O₁₁), 9 to 18 g of agar and 1000 ml (1 liter) of water (solventwater).

Prescribed amounts of components contained in the solid medium describedin Reference 3 are mentioned hereafter.

Prescribed amounts of components in the solid medium composition of theBaird-Parker agar medium (ETGP agar medium, pH 6.8, where10 ml of1%potassium tellurite aqueous solution and 50 ml of 50% egg yolk emulsionare added after sterilization with high pressure steam and beforepouring the medium into plate) are 10 g of peptone, 5 g of meat extract,1 g of yeast extract, 5 g of lithium chloride, 12 g of glycine, 10 g ofsodium pyruvate, 17 g of agar and 1000 ml (1 liter) of purified water(solvent water).

The method for producing a solid medium of the present invention isexplained hereafter.

In the method for producing a solid medium of the present invention,dissolution of components of the solid medium into solvent water andsolidification of the solution can be performed in the same manner as inthe production of usual solid media except that the amount of thesolvent water is larger than its prescribed amount and that water isremoved by drying after the solidification. For example, prescribedamounts of medium components described in literature etc. or newlyidentified as a solid medium composition (defined) can be dissolved insolvent water; after adjusting pH when required, a gelling agent can beadded to the solution; and the mixture can be heated to dissolve thegelling agent, sterilized, poured into a vessel like a dish andsolidified.

The amount of water to be removed is such an amount that the 10minute-average water absorption rate of the solid medium should becomeat least 0.05 ml/minute after the removal of water. Although this amountmay vary depending on the medium composition (especially depending onthe gelling agent), it can be determined by producing media containingsolvent water in various excessive amounts and measuring their 10minute-average water absorption rates in such a manner as described inTest Example 3 mentioned later.

The amount of water to be removed is preferably at least 5% of thesolvent water, more preferably at least 30% of the solvent water.

When water is removed in above-mentioned manner from a solid mediumprepared by using a prescribed amount of solvent water, the amount ofwater contained in the solid medium may be significantly reduced afterthe removal of water, resulting in growth inhibition of microorganismsdue to drying. According to the present invention, the amount of solventwater in the solid medium is determined beforehand to be larger than theprescribed one, so that water is contained in the solid medium in anamount corresponding to the prescribed amount after the removal ofwater. Therefore, the amount of solvent water is larger than theprescribed amount by an amount almost equal to that of water to beremoved. The amount almost equal to that of water to be removed hereinmeans the amount at which, the amount obtained by subtracting the amountof water to be removed from the amount of the total added solvent waterincluding the prescribed amount and the additional amount of water,should become substantially equal to (usually, 97 to 103%) theprescribed amount of water.

Usually, the additional amount of the solvent water is 5 to 150% whenthe amount of solvent water (prescribed amount of solvent water) in acomposition described literature etc., or in a composition newlyidentified as a solid medium composition, is taken as 100%. This amountof solvent water is formulated in addition to the prescribed amount ofwater. This enables the production of the solid medium of the presentinvention which contains a prescribed amount of water and has asufficient water absorption rate, as an end product obtained by removingsolvent water by drying.

When an agar is used as the gelling agent, the concentration of theadded agar is preferably 1.0 to 3% (by weight) when the aforementionedprescribed amount of solvent water is taken as 100%. Further, theheating for dissolution is preferably performed at a temperature of 100°C. or higher, since agar is fully dissolved at such a temperature. Ifthe heating is performed at 121° C. for 15 minutes or more,sterilization can also be attained simultaneously with the dissolution.Sterilization may be performed in a conventional manner by using highpressure steam sterilization method or the like.

When dishes are used as the vessels, the solid medium is preferablypoured into each dish in such an amount that the amount of the solidmedium in each dish after the removal of water by drying should become10 to 30 ml.

Examples of the drying method to remove water from the medium includereduced pressure drying method, vacuum evaporation method, warm airdrying method, infrared drying method, high frequency drying method andso forth. However, the method is not limited to these.

The solid medium of the present invention has 10 minute-average waterabsorption rate of at least 0.05 ml/minute according to the definitionmentioned above. If the solid medium shows water absorption of at least0.5 ml for 10 minutes per one plate of the standard size defined above,it becomes possible to apply a large amount of a sample onto the solidmedium in a short period of time. A solid medium showing the 10minute-average water absorption rate of at least 0.05 ml/minute can beprepared by drying the medium to remove such an appropriate amount ofwater as described above. The amount of medium per exposed area (area incontact with air) of the solid medium can be such an amount that the 10minute-average water absorption rate in the aforementioned range can beobtained. If the amount of the medium is too small, a sufficient 10minute-average water absorption rate may not be obtained.

Since the solid medium of the present invention contains solvent waterin an amount almost equal to the prescribed amount after the removal ofwater, growth inhibition of microorganisms resulting from drying is notobserved on it. This is because a prescribed amount of solvent water ina composition identifying a solid medium is determined so that amicroorganism to be cultured can grow. Alternatively, it is because amedium having a composition that does not cause growth inhibition of amicroorganism to be cultured is selected.

The solid medium preferably has a water content of at least 90%. Suchwater content enables culture of microorganism requiring high watercontent. For example, since growth of a microorganism such asStaphylococcus aureus ATCC 6538P is almost completely inhibited in watercontent lower than 90% as demonstrated in the Test Examples mentionedlater, it is necessary to keep water content of at least 90% in thesolid medium in order to culture the microorganism.

Furthermore, the solid medium of the present invention is suitable forculturing microorganisms that are relatively weak to dryness, butimportant for microorganism tests of foodstuffs and so forth, such asStaphylococcus aureus ATCC 6538P and Bacillus stearothermophilus var.calidolactis NIZO C953, without causing any growth inhibition thereof.As described above, the solid medium of the present invention is driedin a state that it contains solvent water in an amount larger than theprescribed amount. Therefore, it maintains a sufficient water absorptionrate and does not cause growth inhibition due to drying.

In order to make it much easier to understand the method for producing asolid medium of the present invention, it is explained by exemplifying amethod for producing the standard agar medium described in Reference 1mentioned above.

Namely, prescribed amounts of components in the solid medium compositionof the standard agar medium are 2.5 g of yeast extract, 5 g of peptone,1 g of glucose, 15 g of agar and 1000 ml of purified water (solventwater), as described in Reference 1.

Therefore, the component of 2.5 g of yeast extract, 5 g of peptone and 1g of glucose are dissolved in solvent water whose amount is preferably 5to 150% (50 to 1500 ml) larger than the prescribed amount (1000 ml) toobtain a dissolved solution in an amount from about 1050 ml to 2500 ml.The dissolved solution is adjusted to pH 6.8 to 7.2. 15 g of agar isadded to the solution and the mixture is heated at 121° C. for 15minutes by the high pressure steam sterilization method to dissolve andsterilize simultaneously.

The sterilized medium is aseptically poured into dishes in an amount of16 to 38 g per dish, and solidified by cooling. The solidified medium isthen dried by the reduced pressure drying method under a pressure of 10³Pa using a dryer (LABCONCO) to remove solvent water.

By the aforementioned method to remove water by drying, the additionalamount of water is removed to produce a solid medium which has the 10minute-average water absorption rate of at least 0.05 ml/minute.

The solid medium produced by the aforementioned method does not causegrowth inhibition of microorganisms resulting from drying. Moreover, itshows a superior water absorption rate and enables application of alarge amount of a sample in a short period of time and is suitable forquick and accurate measurement tests of microbial numbers.

EXAMPLES

The present invention is explained in more detail by the followingexamples. However, the present invention is not limited to the followingexamples.

Example 1

2.5 g of yeast extract (Oriental Yeast), 5 g of peptone (Difco) and 1 gof glucose (Wako Pure Chemical Industries) were dissolved in 1200 ml ofsolvent water which contains 20% (200 ml) excess water in addition tothe prescribed amount of solvent water (purified water, 1000 ml), whichwas taken as 100%, in a known solid medium composition to obtain about1200 ml of a dissolved solution. The solution was adjusted to pH 7.2 byusing 0.1 mol/l sodium hydroxide solution. 15 g of agar (Ina ShokuhinKogyo) was added to the solution and the mixtures was heated at 121° C.for 15 minutes by using an autoclave (Iwatate Iryo Kikai Seisakusho) toattain dissolution and sterilization at the same time.

The sterilized solid medium was poured into each plastic dish having adiameter of 9 cm (Eiken Kizai) in an amount of 18 g per dish in asterile room, solidified by cooling and dried at a pressure of 10³ Paaccording to the reduced pressure drying method using a drier (LABCONCO)to remove 3 ml of water. As a result, solid medium plates eachcontaining 15 g of the medium were obtained.

The aforementioned removal of water corresponded to removal of the totalamount of the solvent water (200 ml) formulated in addition to theprescribed amount of water, i.e., removal of about 17% (200 ml) of thetotal amount (1200 ml) of the added solvent water (purified water),which was taken as 100%, by drying.

Thus, the water content of the solid medium has been adjusted to about98% by the aforementioned removal of water.

The solid medium (the nutrient agar medium) plates produced as describedabove were tested by the method described later, and they were found tohave the 10 minute-average water absorption rate of 0.15 ml/minute. Ithas been found that the medium is a solid medium capable of applicationof a large amount of a sample in a short period of time and which doesnot show growth inhibition caused by drying, whereby it is suitable forquick and accurate measurement tests of microbial numbers.

Example 2

2.5 g of meat extract (Merck),10 g of peptone (Difco), 75 g of sodiumchloride (Wako Pure Chemical Industries), 10 g of mannitol (Wako PureChemical Industries) and 0.025 g of Phenol Red (Wako Pure ChemicalIndustries) were dissolved in 1066 ml of solvent water which contains6.6% (66 ml) excess water in addition to the prescribed amount ofsolvent water (purified water, 1000 ml) taken as 100%, in a known solidmedium composition, to obtain about 1066 ml of a dissolved solution. Thedissolved solution was adjusted to pH 7.4 by using 0.1 mol/l sodiumhydroxide solution. 15 g of agar (Ina Shokuhin Kogyo) was added to thesolution and the mixture was heated at 121° C. for 15 minutes by usingan autoclave (Iwatate Iryo Kikai Seisakusho) to attain dissolution andsterilization at the same time.

The sterilized solid medium was poured into each plastic dish having adiameter of 9 cm (Eiken Kizai) in an amount of 16 g per dish in asterile room, solidified by cooling and dried at a pressure of 10³ Paaccording to the reduced pressure drying method using a drier (LABCONCO)to remove about 1 ml of solvent water. As a result, solid medium plateseach containing 15 g of the medium were obtained.

The aforementioned removal of water corresponded to removal of the totalamount of the solvent water (66 ml) formulated in addition to theprescribed amount of water, i.e., removal of about 6% (66 ml) of thetotal amount (1066 ml) of the added solvent water (purified water) takenas 100%, was removed by drying.

Therefore, the water content of the solid medium has been adjusted toabout 90% by the aforementioned removal of water.

The solid medium (the mannitol salt agar with egg yolk) plates producedas described above were tested by the method described later, and theywere found to have the 10 minute-average water absorption rate of 0.07ml/minute. It has been found that the medium is a solid medium whichenables application of a large amount of a sample in a short period oftime and which does not show microbial growth inhibition ofStaphyiococcus bacteria caused by drying, whereby it is suitable forquick and accurate measurement tests of microbial numbers.

Example 3

A solid medium having a novel composition was prepared by modifying acomposition of a known solid medium. 10 g of peptone (Difco), 5 g ofmeat extract (Merck) and 2 g of sodium chloride (Wako Pure ChemicalIndustries) were dissolved in 1200 ml of solvent water, which contains33.3% (300 ml) of excess water in addition to a prescribed amount of 900ml, taken as 100%, which was modified to a less amount than theprescribed amount of solvent water (purified water, 1000 ml) in a knownsolid medium composition, to obtain about 1200 ml of a dissolvedsolution. The dissolved solution was adjusted to pH 7.0 by using 0.1mol/l sodium hydroxide solution. 15 g of agar (Ina Shokuhin Kogyo) wasadded to the solution and the mixture was heated at 121° C. for 15minutes by using an autoclave (Iwatate Iryo Kikai Seisakusho) to attaindissolution and sterilization at the same time.

The sterilized solid medium was poured into each plastic dish having adiameter of 9 cm (Eiken Kizai) in an amount of 20 g per dish in asterile room, solidified by cooling and dried at a pressure of 10³ Paaccording to the reduced pressure drying method using a drier (LABCONCO)to remove 5 ml of solvent water. As a result, solid medium plates eachcontaining 15 g of the medium were obtained.

The aforementioned removal water corresponded to removal of the totalamount (300 ml) of the water formulated in addition to the prescribedamount of water, i.e., removal of about 25% (300 ml) of total amount(1200 ml) of the added solvent water(purified water), taken as 100%, bydrying.

Thus, the water content of the solid medium has been adjusted to about96% by the aforementioned removal of water.

The solid medium (nutrient agar medium) plates produced as describedabove were tested by the method described later, and they were found tohave the 10 minute-average water absorption rate of 0.17 ml/minute. Ithas been found that the medium is a solid medium which enablesapplication of a large amount of sample in a short period of time andwhich does not show microbial growth inhibition caused by drying,whereby it is suitable for quick and accurate measurement tests ofmicrobial numbers.

Test Example 1

This test was performed to compare the present invention with the priorart by using results of microbial growth test as indexes.

(1) Preparation of Samples

Each of the following three kinds of samples was prepared inquintuplicate.

-   Sample 1: Solid medium produced in the same manner as in Example 1    of the present invention.-   Sample 2: Solid medium produced in the same manner as in Example 1    of Prior Art 1 except that the type of the medium was changed to the    nutrient agar medium, 15 ml of the medium was poured into each dish,    15 ml of water for restoration (sterilized water) was used, the    solid medium was restored for 5 minutes and excessive water for    restoration (free water) was discarded by decantation.-   Sample 3: Solid medium produced in the same manner as in Example 1    of Prior Art 2 except that the type of the medium was changed to the    nutrient agar medium, 15 ml of the medium was poured into each dish,    15 ml of water for restoration (sterilized water) was used, the    solid medium was restored for 5 minutes and excessive water for    restoration (free water) was discarded by decantation.    (2) Test Method

Microbial growth on each sample was examined by the following testmethod.

As test strains, Staphylococcus aureus ATCC 6538P obtained from theAmerican Type Culture Collection, which is a depository ofmicroorganisms, and Bacillus stearothermophilus var. calidolactis NIZOC953 obtained from the Netherlands Institute for Dairy Research(Nederlands Instituut voor Zuivelonderzoek, NIZO) were used.

Such a dilution of each test strain was prepared that, when 0.1 ml ofthe dilution was applied onto one standard agar medium plate prepared byusing solvent water in the prescribed amount defined in the known solidmedium composition, i.e., using solvent water in the prescribed amountdefined in the known solid medium composition as it was, without anydrying process and incubated at 37° C. for 48 hours, 100 colonies of thetest strain should be obtained.

The solid medium composition of the standard agar medium is composed of2.5 g of yeast extract, 5 g of peptone, 1 g of glucose, 15 g of agar and1000 ml (prescribed amount of solvent water) of purified water.

The dilution of test strain in an amount of 0.1 ml was applied onto oneplate of each sample and incubated at 37° C. for 48 hours, and number ofappeared colonies was determined by visual inspection. The test wasperformed in quintuplicate, and an average of the numbers of colonieswas calculated.

(3) Test Results

The results of this test are as shown in Table 1. As clearly seen fromthe results shown in Table 1, it was found that Sample 1 according tothe present invention is superior to Samples 2 and 3 according to priorart, since Staphylococcus aureus ATCC 6538P and Bacillusstearothermophilus var. calidolactis NIZO C953, which are importantbacteria for microorganism tests of foodstuffs and so forth, could bedetected without any growth inhibition and microbial numbers could bemeasured accurately and quickly with Sample 1.

Based on the above results, it was found that the dried solid media ofthe conventional techniques required a long period of time forrestoration and thus they were not suitable for quick and accuratemeasurement tests of microbial numbers.

In addition, when the test was repeated by changing the type of medium,the amount of medium poured into each dish, the amount of water(sterilized water) for restoration of dried solid medium and therestoration time of dried solid medium (within the range of 1 to 10minutes), almost similar results were obtained.

TABLE 1 Colony number of Colony number of Bacillus Sample Staphylococcusaureus stearothermophilus NIZO No. ATCC 6538P C953 1 102 98 2 35 38 3 4053

Test Example 2

This test was performed in order to determine appropriate water contentin solid medium by using results of microbial growth test as indexes.

(1) Preparation of Samples

Each of the following two kinds of samples was prepared inquintuplicate.

-   Sample 4: Solid medium prepared in the same manner as in Example 2    of the present invention except that solvent water was removed by    drying so that the water content of the solid medium should become    90%-   Sample 5: Solid medium prepared in the same manner as in Example 2    of the present invention except that solvent water was removed by    drying so that the water content of the solid medium should become    80%    (2) Test Method

Microbial growth on each sample was examined in the same manner as thetest method of Test Example 1 mentioned above except that only theStaphylococcus aureus ATCC 6538P mentioned above was used as a teststrain.

Change of water content in the solid medium by drying was monitored interms of weight loss caused by drying. That is, the weight loss bydrying, i.e., so-called water loss (c), was obtained from weight ofmedium before drying (a) and weight of medium after drying (b) inaccordance with the equation of a−b=c. Then, content of remaining waterwas obtained by subtracting the above water loss (c) from the weight ofthe total added solvent water. The water content after drying wasobtained based on the definitions of the remaining water content and theaforementioned water content to monitor the change of water content ofthe solid medium by drying.

The solid medium composition of the mannitol sodium chloride agar mediumis composed of 2.5 g of meat extract, 10 g of peptone, 75 g of sodiumchloride, 10 g of mannitol, 0.025 g of Phenol Red, 15 g of agar and 1000ml (prescribed amount of solvent water) of purified water.

(3) Test Results

The results of this test are as shown in Table 2. As clearly seen fromthe results shown in Table 2, when the water content of the solid mediumwas 80%, so-called less than 90%, growth of Staphylococcus aureus ATCC6538P was almost completely inhibited, which made it impossible to countthe microbial number, thus it was found that a water content of at least90% was necessary to measure the numbers accurately.

In addition, when the test was repeated by changing the type of mediumand the water content variously, almost similar results were obtained.

TABLE 2 Colony number of Staphylococcus aureus ATCC Sample No. 6538P 497 5 0

Test Example 3

This test was performed by using the results of absorption rate test asindexes in order to determine an appropriate amount of solvent water tobe removed (%) when an amount of solvent water described as a solidmedium composition in literature and so forth was taken as 100%.

(1) Preparation of Samples

Each of the following four kinds of samples was prepared inquintuplicate.

-   Sample 6: Solid medium prepared in the same manner as in Example 1    of the present invention except that solvent water was not    formulated in addition to the prescribed amount and removal of    solvent water by drying was not performed-   Sample 7: Solid medium prepared in the same manner as in Example 1    of the present invention except that solvent water was additionally    formulated by 2.5% of the prescribed amount and 2.5% of the total    amount of solvent water was removed by drying.-   Sample 8: Solid medium prepared in the same manner as in Example 1    of the present invention except that solvent water was additionally    formulated by 5% of the prescribed amount and 5% of the total amount    of solvent water was removed by drying.-   Sample 9: Solid medium prepared in the same manner as in Example 1    of the present invention except that solvent water was additionally    formulated by 40% of the prescribed amount and 30% of the total    amount of solvent water was removed by drying.    (2) Test Method

The water absorption rate of each sample was determined by the followingtest method. The determination was performed at 25° C.

The total weight of sample plate (B) was measured, then 10 ml of ionexchanged water was added onto a plate medium (circular shape, outerdiameter 9 cm. inner diameter: 8.6 cm (area: 58.1 cm²), amount ofmedium: 15 g) to start water absorption; water not absorbed wasdiscarded when 10 minutes passed; moisture on a dish wall was wiped off;and the total weight of the sample plate after water absorption (A) wasmeasured. Then, the 10 minute-average water absorption rate wascalculated in accordance with the following equation:Water absorption rate (ml/minute)=(A−B)/10

Further, percentage of solvent water removed by drying (e) was obtainedfrom the total amount of formulated solvent water (d) which was taken as100%, and the water loss (c) monitored and calculated in the same manneras in Test Example 2 in accordance with the following equation:e(%)=c/d×100(3) Test Results

The results of this test are as shown in Table 3. As clearly seen fromthe results shown in Table 3, when at least 5% of solvent water wasremoved by drying the medium, the water absorption rate became at least0.05 ml/minute, and therefore it became possible to apply a large amountof a sample in a short period of time. Thus, it was found that it isnecessary to remove at least 5% of solvent water for quick measurementtests of microbial numbers. Further, when at least 30% of solvent waterwas removed, the water absorption rate became at least 0.2 ml/minute,and therefore it became possible to apply a larger amount of the samplein a short period of time. Thus, it was found that it is preferable toremove at least 30% of solvent water for quick measurement tests ofmicrobial numbers.

In addition, when the test was repeated by changing the type of mediumvariously, almost similar results were obtained.

TABLE 3 10 minute-average water Sample No. absorption rate (ml/minute) 60.02 7 0.04 8 0.06 9 0.2

INDUSTRIAL APPLICABILITY

The advantages of the present invention are as follows.

-   1) The medium of the present invention, which is prepared by the    production method of the present invention, shows a superior water    absorption rate, to enable application of a large amount of sample    in a short period of time, and is suitable for quick measurement    tests of microbial numbers.-   2) Since the solid medium of the present invention, which is    prepared by the production method of the present invention, enables    application of a large amount of sample, it provides high accuracy    for microbial detection and therefore it is suitable for accurate    measurement tests of microbial numbers.-   3) Since the solid medium of the present invention, which is    prepared by the production method of the present invention, does not    show growth inhibition due to drying, it is suitable for accurate    measurement tests of microbial numbers.-   4) A solid medium with a superior water absorption rate which    enables application of a large amount of sample in a short period of    time and suitable for quick and accurate measurement tests of    microbial numbers can be prepared by the production method of the    present invention.

1. A solid agar medium for microorganisms having a water content of atleast 90%, and having a 10 minute-average water absorption rate of atleast 0.05 ml/minute, which is obtainable by a method comprising thesteps of: dissolving components of the solid medium other than solventwater into the solvent water, solidifying the obtained solution, anddrying the solidified medium to remove water, wherein the10-minute-average water absorption rate is calculated for an area of themedium which is about 58.1 cm² with a weight of about 15 gm, and whereinthe amount of the solvent water is larger than a prescribed amount by anamount almost equal to the amount of the water to be removed.
 2. Thesolid agar medium according to claim 1, wherein the amount of theremoved water is at least 5% of the solvent water.
 3. The solid agarmedium according to claim 1, wherein the amount of the removed water tobe removed is at least 30% of the solvent water.
 4. A method forproducing a solid agar medium for microorganisms which comprises thesteps of: dissolving components of the solid agar medium into a solventwater, solidifying the obtained solution, and drying the solidifiedmedium to remove water, wherein water is removed in such an amount thatthe solid medium after the removal of water should have a water contentof at least 90%, and a 10 minute-average water absorption rate of atleast 0.05 ml/minute calculated for an area of the medium which is about58.1 cm² with a weight of about 15 gm, and wherein the amount of solventwater is larger than the prescribed amount by an amount almost equal tothe amount of the removed water.
 5. The method according to claim 4,wherein the amount of the removed water is at least 5% of the solventwater.
 6. The method according to claim 4, wherein the amount of theremoved water is at least 30% of the solvent water.